Concentric zones of active RhoA and Cdc42 around single cell wounds

Rho GTPases control many cytoskeleton-dependent processes, but how they regulate spatially distinct features of cytoskeletal function within a single cell is poorly understood. Here, we studied active RhoA and Cdc42 in wounded Xenopus oocytes, which assemble and close a dynamic ring of actin filaments (F-actin) and myosin-2 around wound sites. RhoA and Cdc42 are rapidly activated around wound sites in a calcium-dependent manner and segregate into distinct, concentric zones around the wound, with active Cdc42 in the approximate middle of the F-actin array and active RhoA on the interior of the array. These zones form before F-actin accumulation, and then move in concert with the closing array. Microtubules and F-actin are required for normal zone organization and dynamics, as is crosstalk between RhoA and Cdc42. Each of the zones makes distinct contributions to the organization and function of the actomyosin wound array. We propose that similar rho activity zones control related processes such as cytokinesis

In Vivo Fluorescent Labeling of Tumor Cells with the HaloTag® Technology

Many fluorescent sensors are currently available for in vitro bio-physiological microscopic imaging. The ability to label cells in living animals with these fluorescent sensors would help translate some of these assays into in vivo applications. To achieve this goal, the first step is to establish a method for selectively labeling target cells with exogenous fluorophores. Here we tested whether the HaloTag® protein tagging system provides specific labeling of xenograft tumors in living animals. After systemic delivery of fluorophore-conjugated ligands, we performed whole animal planar fluorescent imaging to determine uptake in tag-expressing HCT116 xenografts. Our results demonstrate that HaloTag ligands containing red or near-infrared fluorophores have enhanced tumor uptake and are suitable for non-invasive in vivo imaging. Our proof-of-concept results establish feasibility for using HaloTag technology for bio-physiological imaging in living animals

In vivo tracking of human neural progenitor cells in the rat brain using bioluminescence imaging

AbstractBackgroundStem cell therapies appear promising for treating certain neurodegenerative disorders and molecular imaging methods that track these cells in vivo could answer some key questions regarding their survival and migration. Bioluminescence imaging (BLI), which relies on luciferase expression in these cells, has been used for this purpose due to its high sensitivity.New methodIn this study, we employ BLI to track luciferase-expressing human neural progenitor cells (hNPCLuc2) in the rat striatum long-term.ResultsWe show that hNPCLuc2 are detectable in the rat striatum. Furthermore, we demonstrate that using this tracking method, surviving grafts can be detected in vivo for up to 12 weeks, while those that were rejected do not produce bioluminescence signal. We also demonstrate the ability to discern hNPCLuc2 contralateral migration.Comparison with existing methodsSome of the advantages of BLI compared to other imaging methods used to track progenitor/stem cells include its sensitivity and specificity, low background signal and ability to distinguish surviving grafts from rejected ones over the long term while the blood–brain barrier remains intact.ConclusionsThese new findings may be useful in future preclinical applications developing cell-based treatments for neurodegenerative disorders